CN114574441A - 一种黑磷纳米片新的生物学应用 - Google Patents
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Abstract
本发明提供一种黑磷纳米片新的生物学应用,本发明提出一种神经前体细胞培养基,该培养基包括黑磷纳米片,能够提高神经前体细胞的抗氧化能力,从而有效抵抗过氧化氢和糖氧剥离带来的氧化损伤,并提高神经前体细胞在体内或体外应激环境中的存活率。
Description
技术领域
本发明属于生物医药技术领域,尤其涉及一种黑磷纳米片新的生物学应用。
背景技术
脑卒中是脑部血管发生病变引发的恶性疾病,其中缺血性脑卒中占卒中类型的80%以上,具有高死亡率和高致残率的特点。在缺血发生后,及时清除血栓能极大改善患者的预后。而恢复血流灌注后过剩的氧化物质却能引发氧化应激反应,过多的活性氧直接攻击脂质、蛋白质以及核酸,使之发生氧化从而导致细胞的破坏,造成神经组织的二次损伤。氧化应激和一系列过度的炎症反应是缺血性脑卒中后神经功能恢复的主要障碍。
动物模型和临床试验证明移植神经干细胞移植是一种有潜力的治疗脑卒中的方式。神经干细胞移植治疗脑卒中的机制是多模式的,但是大量的证据表明神经干细胞移植对神经功能的改善是轻中度的。其中很关键的原因是神经干细胞移植后大部分细胞不能存活。缺血再灌注造成的氧化损伤不仅对神经组织造成严重损伤,而且极大限制了移植治疗的细胞存活率。因此,提高移植细胞的存活率是提高干细胞移植疗效的关键。
发明内容
本发明旨在至少解决上述现有技术中存在的技术问题之一。为此,本发明第一个方面提出一种神经前体细胞培养基,该培养基包括黑磷纳米片,能够提高神经前体细胞的抗氧化能力,从而有效抵抗过氧化氢和糖氧剥离带来的氧化损伤,并提高神经前体细胞在体内或体外应激环境中的存活率。
本发明的第二个方面提出了一种神经前体细胞的培养方法。
本发明的第三个方面提出了一种神经前体细胞。
本发明的第四个方面提出了一种黑磷纳米片在神经前体细胞培养中的应用
本发明的第五个方面提出了一种黑磷纳米片在制备神经前体细胞培养基中的应用
本发明的第六个方面提出了一种黑磷纳米片在制备神经前体细胞的抗氧化基因表达促进剂中的应用。
本发明的第七个方面提出了一种黑磷纳米片在制备防治神经元损伤药物中的应用。
本发明的第八个方面提出了一种黑磷纳米片在制备防治缺血性脑卒中药物中的应用。
根据本发明的第一个方面,提出了一种神经前体细胞培养基,包括黑磷纳米片。
在本发明的一些实施方式中,所述黑磷纳米片的尺寸为100nm~1000nm,优选为400nm~600nm。
在本发明的一些优选的实施方式中,所述黑磷纳米片的质量浓度为0.1μg/mL~5.0μg/mL。
根据本发明的第二个方面,提出了一种神经前体细胞的培养方法,包括在诱导多能干细胞向神经前体细胞分化的体系中加入黑磷纳米片的步骤。
在本发明的一些实施方式中,所述黑磷纳米片在诱导分化培养的第8天~第14天加入。
在本发明的一些优选的实施方式中,所述黑磷纳米片在所述体系中的浓度为0.1μg/mL~5.0μg/mL。
根据本发明的第三个方面,提出了一种神经前体细胞,所述神经前体细胞根据本发明第二个方面所述的神经前体细胞的培养方法获得。
根据本发明的第四个方面,提出了一种黑磷纳米片在神经前体细胞培养中的应用。
在本发明的一些实施方式中,所述黑磷纳米片的尺寸为100nm~1000nm,优选为400nm~600nm。
在本发明的一些优选的实施方式中,所述黑磷纳米片的质量浓度为0.1μg/mL~5.0μg/mL。
根据本发明的第五个方面,提出了一种黑磷纳米片在制备神经前体细胞培养基中的应用。
在本发明的一些实施方式中,所述黑磷纳米片的尺寸为100nm~1000nm,优选为400nm~600nm。
在本发明的一些优选的实施方式中,所述黑磷纳米片的质量浓度为0.1μg/mL~5.0μg/mL。
根据本发明的第六个方面,提出了治疗有效量的黑磷纳米片在制备神经前体细胞的抗氧化基因表达促进剂中的应用。
根据本发明的第七个方面,提出了治疗有效量的黑磷纳米片在制备防治神经元损伤药物中的应用。
根据本发明的第八个方面,提出了治疗有效量的黑磷纳米片在制备防治缺血性脑卒中药物中的应用。
本发明中使用的“治疗有效量”是指所施用的组合物中所含的本发明的黑磷纳米片的量足以达到预期目的的量,所述预期目的在这种情况下例如是能提高神经前体细胞的抗氧化基因表达;能预防、改善、治疗神经元损失;能预防、改善、治疗缺血性脑卒中。
在一些实施方式中,本发明的黑磷纳米片的治疗有效量可以是,例如0.5μg/mL、1.0μg/mL、1.5μg/mL、2.0μg/mL、2.5μg/mL、3.0μg/mL、3.5μg/mL及其他合适的值。
本发明的有益效果为:
1.本发明中,黑磷纳米片能够提高神经前体细胞的抗氧化能力,从而有效抵抗过氧化氢和糖氧剥离带来的氧化损伤,并提高神经前体细胞在体内或体外应激环境中的存活率。
2.本发明拓展了黑磷纳米材料新的生物学应用,为提高移植干细胞存活率及其在神经系统性疾病中的治疗提高了新思路。
附图说明
下面结合附图和实施例对本发明做进一步的说明,其中:
图1为本发明实施例1黑磷纳米片的表征图,其中(a)为黑磷纳米片透射电镜图片和(b)为黑磷纳米片的粒径分布图。
图2为本发明实施例2神经前体细胞培养流程示意图及神经前体细胞的检测结果,其中,(a)是从iPSC分化为NPC的流程图;(b)为对照组NPC和经过黑磷纳米片处理的NPC的细胞形态图片;(c)为不同浓度黑磷纳米片与从iPSC向NPC分化的P1代细胞共培养5天的细胞毒性检测结果图。
图3为实施例3中NPC细胞抗氧化基因表达结果图,其中,(a)为qPCR检测NPC的谷氨酸转运蛋白及相关抗氧化基因的表达;(b)为WB检测Nrf2、GCLM、HMOX1、GPX1和NQO1蛋白的表达;(c)为图(b)的定量结果。
图4为实施例4中NPC对氧化损伤抵抗作用效果图,其中,(a)为有无黑磷纳米片处理的神经前体细胞经过氧化氢处理6小时后活死细胞染色结果;(b)为有无黑磷纳米片处理的神经前体细胞经过无糖培养基处理6小时后活死细胞染色结果。
图5为实施例5中神经前体细胞对神经元的氧化损伤的保护作用效果图,其中,(a)为过氧化氢诱导神经元氧化损伤时,不同培养基处理的神经元活化的凋亡蛋白的免疫荧光染色;(b)是图(a)的定量结果。
图6为实施例6中NPC对脑内保护作用效果图,其中,(a)为细胞移植3天后大鼠海马区所检测到的人细胞核以及巢蛋白的免疫荧光图;(b)是图(a)的定量结果。
图7为实施例7中神经前体细胞对大鼠缺血性脑卒中的治疗效果,其中,(a)为细胞移植后第3天大鼠的神经功能评分;(b)为ELISA试剂盒检测细胞移植后18天大鼠损伤半脑组织匀浆上清中MDA的含量结果;(c)为细胞移植18天后大鼠海马区的NEUN和GFAP的免疫荧光图片;(d)是图(c)的定量结果。
附图标记:“ns”表示“无显著性差异”“*”表示“p<0.05”;“**”表示“p<0.01”;“***”表示“p<0.001”。
具体实施方式
以下将结合实施例对本发明的构思及产生的技术效果进行清楚、完整地描述,以充分地理解本发明的目的、特征和效果。显然,所描述的实施例只是本发明的一部分实施例,而不是全部实施例,基于本发明的实施例,本领域的技术人员在不付出创造性劳动的前提下所获得的其他实施例,均属于本发明保护的范围。
实施例1
本实施例制备了一种黑磷纳米片,具体过程为:
将≦30mg的黑磷粉末分散于50mL N-甲基吡咯烷酮溶液中,在冰水浴中用探头式超声破碎仪(参数设置:超声5s停5s,50%功率)进行破碎24h,离心取1000~4000r段沉淀;用超纯水洗涤2次。
浓度测试:再次将产物分散至适量纯水中。用紫外分光光度计测量波长为460nm的吸光度来测定黑磷纳米片的浓度。使用前水浴超声分散。
表征:使用透射电镜图和粒径仪测量黑磷纳米片的尺寸。结果如图1所示,其中图1(a)为黑鳞纳米片的透射电镜图,标尺:2μm;图1(b)为黑鳞纳米片的粒径分布图。
从图1可看出,黑磷纳米片的尺寸集中在500nm左右。
实施例2
本实施例培养了一种神经前体细胞,具体过程为:
图2(a)示出了从iPSC分化为NPC的流程图,黑磷纳米片在分化第8天加入。mTeSR为iPSC培养基;NIM1为NPC诱导培养基1,具体成分为50%DMEM/F12,50%Neurobasal,1x N2,1x B27,1X GlutaMAX,10ng/mL hLIF,4μM CHIR99021,3μM SB431542,2μM Dorsomorphin,and 0.1μM Compound E;NIM2为NPC诱导培养基2,具体成分为50%DMEM/F12,50%Neurobasal,1x N2,1x B27,1X GlutaMAX,10ng/mL hLIF,4μM CHIR99021,3μM SB431542,and 0.1μM Compound E;NSMM为NPC的维持培养基,具体成分为50%DMEM/F12,50%Neurobasal,1x N2,1x B27,1X GlutaMAX,10ng/mL hLIF,3μM CHIR99021,and 2μMSB431542。如图2(a)所示,在从诱导人源诱导多能干细胞(iPSC)向神经前体细胞(NPC)方向分化的第8天,即进行单细胞传代的时候,用含有黑磷纳米片的基质胶进行铺板,并把还未完全分化为神经前体细胞的干细胞消化为单细胞在预铺胶的细胞培养皿上传代。大约两天进行一次传代,P3代细胞形态趋于稳定。
观察黑鳞纳米片对NPC细胞形态的影响,对照组和经黑磷纳米片处理组(BP)结果如图2(b)所示,标尺:100μm。
从图2(b)可看出,经黑磷纳米片处理过的神经前体细胞间联系紧密,聚团生长。
此外,还检测了不同浓度黑鳞纳米片与P1代iPSC-NPC细胞共培养5天后的细胞存活率,结果如图2(c)所示。
从图2(c)可看出,在0~20μg/mL的浓度范围内,黑磷纳米片对iPSC-NPC的细胞毒性呈浓度依赖性。
实施例3
本实施例对比了正常对照组与黑磷纳米片组对NPC细胞抗氧化基因表达的影响,具体过程为:
按照实施例2的分化方法,收集P3代的NPC细胞(正常对照组和黑磷处理组),提取了总RNA,之后进行了qPCR实验。结果如图3所示。其中,图3(a)为qPCR检测NPC的谷氨酸转运蛋白及相关抗氧化基因的表达;图3(b)为WB(蛋白印迹)检测抗氧化相关蛋白的表达及图3(c)定量。其中GAPDH为总蛋白的内参蛋白,而Lamin-B1为核蛋白的内参蛋白。C-Nrf2为细胞质的Nrf2蛋白,N-Nrf2为细胞核内的Nrf2蛋白。
从图3(a)基因表达结果可看出,黑磷纳米片上调了NPC的半胱氨酸/谷氨酸逆向转运体系统(SLC7A11/SLC3A2)以及下游的谷氨酸半胱氨酸连接酶修饰亚基(GCLM)。此外抗氧化基因包括红细胞衍生核因子2样蛋白2(NFE2L2),血红素加氧酶1(HMOX1),NAD(P)H:醌氧化还原酶1(NQO1)和谷胱甘肽过氧化酶1(GPX1)的表达也升高。
从图3(b)、图3(c)蛋白表达结果可看出,黑磷纳米片促进了NPC的Nrf2蛋白(由NFE2L2基因编码)的核转录,增强了GCLM,GPX1,HMOX1和NQO1的蛋白表达量,与qPCR结果一致。
实施例4
本实施例研究黑磷纳米片处理过的NPC能否对氧化损伤有更好的抵抗作用,具体过程为:
将经过含有2.5μg/mL黑磷纳米片的基质胶(PBS)培养了4天的NPC按适合细胞密度重新传代,经过24h后,用浓度为1mM的过氧化氢溶液处理6h制造了细胞的氧化损伤,如图4(a)所示,标尺:100μm,采用Calcein-AM/PI双染的方法染色活细胞(绿色)和死细胞(红色),发现黑磷纳米片处理过的NPC能更有效地抵御过氧化氢造成的氧化损伤。
此外,用无糖培养基来模拟细胞的糖剥离环境,比对正常培养基和无糖DMEM培养基下对照和BP处理组的NPC细胞状态,如图4(b)所示,标尺:100μm,采用Calcein-AM/PI双染的方法染色活细胞(绿色)和死细胞(红色),发现黑磷纳米片处理过的NPC能更有效地抵御糖剥离造成的损伤。
综上所述,黑磷纳米片提高神经前体细胞体外氧化应激环境中的存活率。
实施例5
本实施例研究了黑磷纳米片处理过的神经前体细胞对神经元的氧化损伤的保护作用,具体过程为:
提取了NPC的条件培养基,即通过收集培养了48h的P3代NPC的培养基上清(对照组细胞正常培养,黑磷纳米片组在基质胶中加入2.5μg/mL的黑磷纳米片)。通过4000g/r离心30min去除细胞碎片,然后用3kDa的超滤管浓缩上清,然后进行蛋白浓度定量,最后使用以100μg/mL的终浓度添加在培养基(PBS)中。实验分为4组,对照组;H2O2组(浓度为1mM);H2O2+NCM组;H2O2+BP-NCM组。H2O2:过氧化氢。NCM:来源于NPC的条件培养基。BP-NCM:来源于黑磷处理过的NPC的条件培养基。经过不同组别处理24h后对细胞进行活化的凋亡蛋白(caspase-3)的免疫荧光染色-红色,细胞核被染成蓝色,来评估神经元的凋亡情况。
如图5(a)所示,标尺:100μm,过氧化氢诱导神经元氧化损伤时,不同培养基处理的神经元结果证明NPC的条件培养基能够有效减少神经元的凋亡,进一步根据凋亡蛋白的免疫荧光强度定量结果(图5(b)),H2O2+BP-NCM组比起H2O2组相对免疫荧光强度减少了近10倍,证明黑磷纳米片处理过的NPC产生的条件培养基对神经元的氧化损伤有更好的保护作用。
实施例6
本实施例进一步研究NPC的脑内保护作用,具体过程为:
建立大鼠的中动脉栓塞(MCAO)模型,MCAO模型用前端包被有多聚赖氨酸的尼龙线栓从大鼠的颈内动脉插入到中动脉,阻断血流60min后拔出线栓,模拟脑部短暂缺血后造成的缺血再灌注损伤。6h以后进行细胞移植。假手术组(Sham)除不插入线栓和注射以外,其余操作相同。所移植细胞为经过正常培养(NPC组)和在基质胶中加入2.5μg/mL的黑磷纳米片培养(NPC(BP-处理)组)的P3代NPC细胞。细胞注射量为每只5x105个,体积为5μL,PBS组每只注射5μL PBS。3日后牺牲部分大鼠检测移植细胞的存活情况。
如图6(a)所示,标尺:100μm,通过特异性针对人细胞核(hN)以及人源神经干细胞标记蛋白-巢蛋白(NESTIN)的荧光染色-阳性细胞被染成绿色,来检测移植NPC在大鼠海马区的存活情况。从图6(b)可看出,黑磷处理过的NPC的细胞存活数要高于单纯的NPC移植组,通过imageJ统计单位面积内BP处理过的NPC细胞存活数要比NPC组多20个左右。
实施例7
本实施例进一步研究黑磷纳米片处理过的神经前体细胞对大鼠缺血性脑卒中的治疗效果。
在细胞移植到MCAO大鼠的第3天,由不知情的其他实验人员根据Longa评分法对大鼠进行了神经功能评分。实验结果如图7(a)所示,PBS组和NPC组大鼠评分集中在2分和3分,而黑磷处理过的NPC组则集中在1分和2分。结果显示移植了黑磷纳米片处理过的NPC的大鼠有更好的行为学表现。
丙二醛(MDA)作为氧自由基攻击细胞膜发生脂质过氧化反应的产物之一,其水平能反应组织的过氧化程度。因此为了检查脑部氧化损伤程度,在细胞移植后的第18天,研磨了大鼠的损伤半脑并且通过离心(15000g/min,10min)取上清液,进一步用ELISA试剂盒检测了大鼠脑组织中丙二醛的含量。如图7(b)所示,Sham组MDA含量为3.56nmoL/mL,PBS组MDA含量为3.94nmoL/mL,NPC组MDA含量为3.84nmoL/mL,NPC(BP处理)组MDA含量为3.67nmoL/mL,与Sham组(假手术组)相比,MCAO发生后组织存在不同程度的脂质过氧化情况,但是经黑磷纳米片处理过的NPC组相对于PBS组和NPC组MDA含量最低,说明经黑磷纳米片处理过的NPC组有最为显著的抗氧化效果,这与在体外所观察到的结果是相符合的。
星形胶质细胞是脑组织中主要的炎症细胞之一,它的异常激活反映了大脑中的炎症状态。本实施还检测了细胞移植18天后MCAO大鼠脑部星形胶质细胞的数量和活化情况。如图7(c)所示,标尺:100μm,通过对大鼠海马区同时复染了NEUN(神经元标记物,红色)和GFAP(胶质纤维酸性蛋白:星形胶质细胞标记物,绿色),细胞核被DAPI染为蓝色,并且从左到右依次列出了大鼠的齿状回区,CA3/CA2,CA1区和大脑皮层区。可以清楚地看到PBS组四个区域都聚集了大量的星形胶质细胞。组织破损形成明显的胶质瘢痕,伴随着神经元数量锐减。NPC移植组都有效地减少了星形胶质细胞的聚集,但经黑磷纳米片处理过的NPC组效果更为显著。定量情况如图7(d)所示,各组GFAP/DAPI阳性率结果为:Sham组为12.73%,PBS组为48.17%,NPC组为35.77%,NPC(BP-处理)组为21.13%;各组NEUN/DAPI阳性率结果为:Sham组为85.38%,PBS组为31.70%,NPC组为57.21%,NPC(BP-处理)组为74.10%。通过定量情况更能确定黑磷纳米片处理过的神经前体细胞对大鼠缺血性脑卒中有更好的治疗效果。
上面对本发明实施例作了详细说明,但是本发明不限于上述实施例,在所属技术领域普通技术人员所具备的知识范围内,还可以在不脱离本发明宗旨的前提下作出各种变化。此外,在不冲突的情况下,本发明的实施例及实施例中的特征可以相互组合。
Claims (10)
1.一种神经前体细胞培养基,其特征在于:包括黑磷纳米片。
2.根据权利要求1所述的神经前体细胞培养基,其特征在于:所述黑磷纳米片的质量浓度为0.1μg/mL~5.0μg/mL。
3.一种神经前体细胞的培养方法,其特征在于:包括在诱导多能干细胞向神经前体细胞分化的体系中加入黑磷纳米片的步骤。
4.根据权利要求3所述的神经前体细胞的培养方法,其特征在于:所述黑磷纳米片在诱导分化培养的第8天~第14天加入。
5.一种神经前体细胞,其特征在于:所述神经前体细胞为权利要求3或4所述的神经前体细胞的培养方法获得。
6.一种黑磷纳米片在神经前体细胞培养中的应用。
7.一种黑磷纳米片在制备神经前体细胞培养基中的应用。
8.治疗有效量的黑磷纳米片在制备神经前体细胞的抗氧化基因表达促进剂中的应用。
9.治疗有效量的黑磷纳米片在制备防治神经元损伤药物中的应用。
10.治疗有效量的黑磷纳米片在制备防治缺血性脑卒中药物中的应用。
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